It is the goal of this research plan to provide a definitive study of the molecular role of metal ion binding (Ca++, Mg++, Eu+++, Eu++) to the blood clotting proteins prothrombin fragment 1 and prothrombin 1-39 from human and bovine sources. Both experimental and theoretical techniques will be used. Experimentally our plan is to: (1) conduct as series of conditions - consistent binding measurements by the techniques of equilibrium dialysis, ultrafiltration and atomic absorption; (2) prepare, characterize and utilize Eu++ as a potential luminescent Ca++ probe; (3) utilize monoclonal antibody binding to specific metal-ion induced conformations of prothrombin fragment 1 so as to determine affinity constants and thus make possible quantitative comparison (between metals) of metal ion induced states. Theoretically our plan is to (1) utilize the binding curves determined experimentally to distinguish between possible cooperativity models; (2) determine by ab initio quantum mechanical methods how Ca++ (and Mg++) binds at the molecular level to Gla and Gla-Gla and their analogs; (3) carry out energy minimization calculations for prothrombin 1-39 so as to find the low energy forms in the presence of metal binding. The relationship between the theoretical concept of cooperativity and experimental binding data is at the core of understanding the energetics and dynamics of protein function. Our overall goal is to clarify this relationship by a study on an important health-related system that we feel can be quantitatively defined.